This invention relates to the use of agents which bind to dipeptidyl peptidase IV (DPIV, also known as CD26) for the stimulation of hematopoietic cells in vitro
Bone marrow transplantation is widely used with patients undergoing high dose chemotherapy or radiation therapy. The dose limiting side effects of chemotherapy and radiation therapy are their deleterious effects on hematopoietic cells through destruction of the bone marrow cells which are the precursor cells for all hematopoietic cells. This damage to the marrow results in myelosuppression or myeloablation, rendering patients susceptible to opportunistic infections for a prolonged period of time. Bone marrow transplantation involves the infusion of early bone marrow progenitor cells that have the ability to re-establish the patients"" hematopoietic system, including the immune system. Transplantation decreases the time normally required for the restoration of the immune system after chemotherapy or radiation therapy and, thus, the time of risk for opportunistic infections.
Bone marrow cells contain totipotent stem cells which give rise to hematopoietic cells of all lineages including the lymphoid, myeloid and erythroid lineages. Stem cells have the ability to renew themselves as well as to differentiate into progenitor cells of all hematopoietic lineages. Progenitor cells retain the ability to proliferate and give rise to differentiated cells of all lineages. Differentiated cells lose the ability to proliferate and exhibit morphological characteristics specific for their lineages (such as macrophages, granulocytes, platelets, red blood cells, T cells and B cells). Stem cells and progenitor cells express CD34 on their surface while differentiated cells do not. Bone marrow includes stem cells as well as progenitor cells of the lymphoid (T and B cells), myeloid (granulocytes, macrophages) and erythroid (red blood cells) lineages.
For use in bone marrow transplants, hematopoietic precursor cells can be derived either from the cancer patient (autologous transplant) or from a histocompatible donor (allogeneic donor). These cells can be isolated from bone marrow, peripheral blood or from umbilical cord blood. In all cases, cells are harvested before chemotherapy or radiation therapy. The number of progenitor cells that can be harvested at one time is small and, in many cases, is not sufficient for a successful transplant. Accordingly, several methods have been developed to expand, in vitro, bone marrow cells or progenitor cells obtained from blood aphereses or from umbilical cord blood.
The ability to expand these cells has helped advance bone marrow transplant technology as a viable adjunct therapy for cancer treatments that involve high doses of chemotherapy and/or irradiation. However, the existing methods for hematopoietic cell expansion require the addition of appropriate cytokines to permit the in vitro expansion of hematopoietic stem cells. The high cost of such growth factors has adversely affected the ability of those skilled in the art to expand hematopoietic cells in vitro for transplantation or other purposes. Accordingly, a need exists to develop new methods for expanding hematopoietic cells in vitro which do not require exogenously added cytokines to support cell growth and differentiation.
The present invention provides methods and compositions for stimulating the growth and differentiation of hematopoietic cells in vitro. Advantageously, the methods of the invention do not require the addition of exogenously added cytokines to support the stimulation of hematopoietic cells in vitro. Accordingly, the methods and compositions of the invention are useful for increasing the number of hematopoietic cells in vitro and/or causing the differentiation of early progenitor cells. Increasing the number and/or differentiation of hematopoietic cells in culture permits the characterization of such cells in culture under a variety of conditions, as well as the use of such cultured cells for the production of recombinant or naturally occurring molecules therefrom in vitro. In addition, the stimulated hematopoietic cells of the invention are useful for the treatment of disorders that are characterized by a reduced number of hematopoietic cells or their precursors in vivo. Such conditions occur frequently in patients who are immunosuppressed, for example, as a consequence of chemotherapy and/or radiation therapy for cancer.
The novelty of the invention is based, at least in part, on the discovery that inhibitors of dipeptidyl peptidase type IV (xe2x80x9cDPIVxe2x80x9d) are useful for stimulating the growth and differentiation of hematopoietic cells in the absence of exogenously added cytokines or other growth factors or stromal cells. This discovery contradicts the dogma in the field of hematopoietic cell stimulation which provides that the addition of cytokines or cells that produce cytokines (stromal cells) is an essential element for maintaining and stimulating the growth and differentiation of hematopoietic cells in culture. (See, e.g., PCT Intl. Application No. PCT/US93/017173, published as WO94/03055).
According to one aspect of the invention, a method for stimulating hematopoietic cells to grow and differentiate in vitro is provided. The method involves: (1) contacting the hematopoietic cells with a sufficient amount of an inhibitor of a dipeptidyl peptidase type IV to increase the number and/or differentiation of hematopoietic cells when the cells are cultured in the presence of the inhibitor relative to the number and differentiation of hematopoietic cells that are present in a control culture that is not contacted with the inhibitor but is otherwise subjected to the same culture conditions as the hematopoietic cells which are cultured in the presence of the inhibitor; (2) culturing the hematopoietic cells in the presence of the inhibitor and in the absence of exogenously added cytokine under conditions and for a time sufficient to increase the number of hematopoietic cells and/or the differentiation of such cells relative to the number of hematopoietic cells that were present in the control culture; (3) culturing the hematopoietic cells in the presence or absence of stromal cells, and (4) culturing stromal cells in the presence of the DPIV inhibitor. In general, increasing the number of hematopoietic cells refers to increasing the number of cells by at least approximately 2-fold relative to the number of hematopoietic cells that are present when the cells initially are contacted with the inhibitor. In general, the number of cells that are present in a control culture that is not contacted with the inhibitor but is otherwise identically treated is approximately the same as the initial number of cells in the culture prior to contact with the inhibitor. Preferably, the number of hematopoietic cells are increased at least approximately 4-fold, 10-fold, 20-fold or, most preferably, at least 100-fold relative to the number of hematopoietic cells that are present when the hematopoietic cells initially are contacted with the inhibitor.
As used herein, hematopoietic cells includes hematopoietic stem cells, primordial stem cells, early progenitor cells, CD34+ cells, early lineage cells of the mesenchymal, myeloid, lymphoid and erythroid lineages, bone marrow cells, blood cells, umbilical cord blood cells, stromal cells, and other hematopoietic precursor cells that are known to those of ordinary skill in the art.
As used herein, an inhibitor of dipeptidyl peptidase type IV (xe2x80x9cDPIVxe2x80x9d) generally refers to a molecule which inhibits the functional activity of the DPIV. Accordingly, the inhibitors of the invention include inhibitors of the enzymatic activity of the dipeptidyl peptidase type IV. Preferably, the inhibitors of the enzymatic activity of DPIV associate with the active site of DPIV by covalently bonding thereto or by forming an ionic interaction therewith. Such inhibitors include competitive inhibitors of DPIV, such as transition state analogs of DPIV, and non-competitive inhibitors of DPIV, such as fluoroalkylketones. Inhibitors of DPIV also include non-competitive inhibitors of DPIV which selectively bind to DPIV (covalently or via ionic interactions) at a site on the DPIV protein other than the active site and, thereby, inhibit the enzymatic activity of the DPIV. Such non-competitive inhibitors are one category of binding molecules which selectively bind to DPIV and have the ability to stimulate hematopoietic cells or thymocytes in vitro. Other binding molecules which selectively bind to DPIV and have the ability to stimulate hematopoietic cells include monoclonal antibodies, polyclonal antibodies and fragments of the foregoing which are capable of: (1) binding to DPIV, and (2) stimulating hematopoietic cells and/or thymocytes in vitro. The inhibitors of DPIV that are useful in the context of the present invention may be immobilized or insoluble form. In general, the foregoing inhibitors can be monovalent, bivalent, or multivalent. (See e.g., U.S. Ser. Nos. 08/671,756 and 08/837,305, entitled xe2x80x9cMultivalent Compounds for Crosslinking Receptors and Uses Thereofxe2x80x9d for a description of dimers and other conjugates of DPIV inhibitors.) The immobilized DPIV inhibitor may be immobilized to a variety of immobilization structures including conventional culture vessels (e.g., stirring flasks, stirred tank reactors, air lift reactors, suspension cell reactors, cell adsorption reactors and cell entrapment reactors, petri dishes, multi well plates, micro titer plates, test tubes, culture flasks, bags and hollow fiber devices, and cell foam. Such immobilization structures preferably are formed of materials including, for example, polystyrene, polypropylene, acrylate polymers, nylon, cloth, nitrocellulose, agarose, sepharose, and so forth.
According to this method of the invention, the hematopoietic cells in an immobilization structure or in an alternative cell culturing device containing soluble DPIV inhibitor are contacted with a sufficient amount of an inhibitor of DPIV to increase the number of hematopoietic cells and/or to cause the differentiation of such cells when the cells are cultured in the presence of the inhibitor. In general, the determination of an increase in the number of hematopoietic cells and/or their state of differentiation is assessed using conventional methods known to those of ordinary skill in the art. An important advantage of the present invention is that the cultured hematopoietic cells can be caused to differentiate and/or increase in number in the absence of exogenously added cytokines. By providing a method for stimulating hematopoietic cells in the absence of exogenously added cytokines, the invention provides substantial cost savings to the culturing of such cells, as well as advantageously reducing the likelihood of contamination of such cell cultures by eliminating what applicants have discovered is no longer an essential agent for stimulating hematopoietic cells in culture.
According to another aspect of the invention, an apparatus is provided for practicing the methods of the invention. The apparatus includes a container and an inhibitor of DPIV contained therein or attached thereto. Preferably, the container is a sterile container which is selected from any of the foregoing cell culture containers known to those of ordinary skill in the art. The inhibitor of DPIV is contained in the container in soluble or immobilized form or directly attached to the internal surface of the container. For example, the inhibitor of DPIV can include magnetic particles to which are attached one or more different inhibitors of DPIV. In addition to containing the immobilized or soluble DPIV inhibitor, the container optionally includes one or more growth media components for cell culture. Such components are known to those of ordinary skill in the art.
According to yet another aspect of the invention, a kit for stimulating hematopoietic cells in culture is provided. The kit contains the apparatus described above and instructions for using the apparatus to stimulate hematopoietic cells in vitro.
According to still another aspect of the invention, a method for stimulating hematopoietic cells and expanding antigen-specific T cells in vitro is provided. The stimulating and expansion steps can be performed concurrently or sequentially. Three embodiments of this method are described below to illustrate this method. In general, the embodiments differ from one another in the selection of the hematopoietic cells that are stimulated in vitro. In each embodiment, the culturing step(s) can be performed in the presence or absence of added cytokines or stromal cells. The preferred heteroconjugates that are used in each embodiment contain a tumor-specific antigen or a pathogen-specific antigen conjugated to a DPIV inhibitor of the invention.
The first embodiment of the method for obtaining antigen-specific T cells involves stimulating bone marrow cells in culture. The bone marrow cells in culture may include a mixture of cells; however, preferably, the bone marrow cells in culture are isolated CD34+ cells or isolated stem cells. According to this embodiment, the method involves: (1) culturing the bone marrow cells in the presence of a sufficient amount of a DPIV inhibitor (e.g., a DPIV monomer and/or homoconjugate) to expand the number of early T lineage cells in culture; and (2) culturing the early T lineage cells with a sufficient amount of a heteroconjugate containing an inhibitor of a DPIV inhibitor attached to an antigenic peptide (e.g., a tumor- or pathogen-specific antigen) to expand the number of antigen-specific T cells in the culture. Step (2) can be performed in the presence or absence of specific antigen. Steps (1) and (2) can be performed concurrently or sequentially. In general, the number of antigen-specific T cells is compared to a control culture of bone marrow cells that is treated as described in steps (1) and (2) with the exception that the control culture is not contacted with the heteroconjugate. At each step, the cells are cultured in the presence of the DPIV inhibitor or heteroconjugate for a time sufficient to increase the number of early T lineage cells and to expand the number of antigen-specific T cells, respectively, relative to the numbers of such cells that are present in the control culture.
The second embodiment is directed to stimulating umbilical cord blood cells in culture. This embodiment involves: (1) culturing the umbilical cord blood cells in the presence of a sufficient amount of a DPIV inhibitor (e.g., a DPIV monomer and/or homoconjugate) to expand the number of early T lineage cells in culture; and (2) culturing the early T lineage cells with a heteroconjugate containing an inhibitor of a DPIV inhibitor attached to an antigenic peptide (e.g., a tumor- or pathogen-specific antigen) to expand the number of antigen-specific T cells that are present in the culture. Step (2) can be performed in the presence or absence of the specific antigen. Steps (1) and (2) can be performed concurrently or sequentially. In general, the number of antigen-specific T cells is compared to a control culture of umbilical cord blood cells that is treated as described in steps (1) and (2) with the exception that the control culture is not contacted with the heteroconjugate. At each step, the cells are cultured in the presence of the DPIV inhibitor or heteroconjugate for a time sufficient to increase the number of early T lineage cells and to expand the number of antigen-specific T cells, respectively, relative to the numbers of such cells that are present in the control culture.
The third embodiment is directed to stimulating peripheral blood stem cells in culture. This embodiment involves: (1) culturing the peripheral blood stem cells in the presence of a sufficient amount of a DPIV inhibitor (e.g., a DPIV monomer and/or homoconjugate) to expand the number of T cells in culture; and (2) culturing the T cells with a sufficient amount of a heteroconjugate containing an inhibitor of a DPIV inhibitor attached to an antigenic peptide (e.g., a tumor- or pathogen-specific antigen) to expand the number of antigen-specific T cells in the culture. Step (2) can be performed in the presence or absence of the specific antigen. Steps (1) and (2) can be performed concurrently or sequentially. In general, the number of antigen-specific T cells is compared to a control culture of peripheral blood stem cells that is treated as described in steps (1) and (2) with the exception that the control culture is not contacted with the heteroconjugate. At each step, the cells are cultured in the presence of the DPIV inhibitor or heteroconjugate for a time sufficient to increase the number of T cells and to expand the number of antigen-specific T cells, respectively, relative to the numbers of such cells that are present in the control culture. Alternatively, because peripheral blood is known to contain T cells, it is possible to expand the number of antigen-specific T cells in culture without the stimulation step (1), i.e., the method for expanding the number of antigen-specific T cells involves culturing the peripheral blood cells with a sufficient amount of a heteroconjugate containing an inhibitor of a DPIV inhibitor attached to an antigenic peptide (e.g., a tumor- or pathogen-specific antigen) to expand the number of antigen-specific T cells in the culture. This step can be performed in the presence or absence of the specific antigen.
These and other aspects of the invention, as well as various advantages in utilities will be more apparent with reference to the drawings and detailed description of the invention.